1. Field of the Invention
The present invention relates to glycopolymers and to methods and compositions for synthesizing glycopolymers. More particularly, it relates to use of Endo-.beta.-N-acetylglucosaminidase from Arthrobacter protophormiae, to produce neoglycoconjugates containing high-mannose type chains.
2. Background Information
Carbohydrates possess important biological functions, such as cell-cell recognition (Wassarman, 1991; Patankar et al, 1993; and Lasky et al., 1992), lectin binding (Lee, 1988; and Lee et al., 1991, Pure & Appl Chem.), viral infection (Glick et al., 1991; and Toogood et al., 1991). Studies of carbohydrate functions require structurally well-defined and highly pure compounds which are usually difficult to obtain from the natural sources. Consequently, synthesis and construction of neoglycoconjugates (proteins, lipids and other types of compounds that have been derivatized with mono- or oligosaccharides) have rapidly gained attention during the past decade (Lee, 1994). Chemical syntheses of neoglycoconjugates have been aggressively developed, but they usually involve multiple, laborious steps. Synthesis of high mannose type oligosaccharides has proven to be especially difficult, even with enzymatic methods.
Endo-.beta.-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) is a glycosidase which performs both hydrolytic and transglycosylation functions. This enzyme cleaves the glycosidic bond in the core GlcNAc.beta.1,4GlcNAc residues of high mannose type and hybrid type N-linked sugar chains in glycoprotein (Takegawa et al., 1989) and also transfers oligosacharide to some mono- and disaccharides (Takegawa et al., 1991a, 1991b). (High mannose type compounds are compounds with only 2-acetylglucosamine residues immediately adjacent to the asparagine, with the remainder of the chain being branched and usually consisting of mannose only, although further modifications with xylose and fucose are sometimes seen. A complex type compound is one consisting of N-acetylglucosamine, galactose, and sometimes fucose and sialic acids. A hybrid type compound is a hybrid of the two.)
The efficiency of the transglycosylation reaction can be markedly increased by addition of organic solvents such as acetone, dimethyl sulfoxide (DMSO) and N,N-dimethyl formamide (DMF), to the reaction solution. For example, when transglycosylation activity of Endo-A is measured using Man.sub.9 -GlcNAc.sub.2 Asn as the donor and GlcNAc as acceptor, the ratio of transglycosylation to hydrolysis is 1:2 in aqueous medium, but when 30% acetone is added, transglycosylation will be performed to near completion. This characteristic makes it possible to synthesize novel glycosides and neoglycoconjugates with high efficency and purity.
Using this method we have synthesized several functional intermediates for neoglycoconjugates, one of which was converted into a glycopolymer with pendant Man.sub.9 GlcNAc.sub.2 chains. The glycopolymer thus prepared displays a drastically greater inhibition of binding by mannose-binding protein from liver over the monomer oligosaccharide.